The Arabidopsis YUCCA1 Flavin Monooxygenase Functions in the Indole-3-Pyruvic Acid Branch of Auxin Biosynthesis W AnnaN.Stepanova,a JeongaYun,a LindaM.Robles,a OndrejNovak,b,c WenrongHe,d HongweiGuo,d KarinLjung,b and Jose M. Alonsoa,1 a Department of Genetics, North Carolina State University, Raleigh, North Carolina 27695 b Umea˚ Plant Science Centre, … These crystals belong to the same space group (P1), but the unit cell dimensions differ from those obtained with the enzyme–FAD complex crystals, giving four molecules per unit cell. Diffraction-quality crystals were obtained by sitting drop-vapor diffusion against a reservoir containing 20% PEG 4000 and 0.1 M sodium citrate buffer (pH 5.8) with 1,6-diaminohexane as additive. The FMO family of genes is conserved across all phyla that have been studied so far, therefore some form of the FMO gene family can be found in all studied eukaryotes. Another difference is in the brain, where adults mostly express FMO2 and fetuses mostly express FMO1. These non-xenobiotic roles of plant FMO's suggest that other FMO functions could be identified in non-plant organisms. [17], Unlike mammals, yeast (Saccharomyces cerevisiae) do not have several isoforms of FMO, but instead only have one called yFMO. It … Online ISSN 1091-6490. Flavin mixed function oxidase. They are divided into three classes, namely FMOs, N-hydroxylating monooxygenases, and Baeyer–Villigar monooxygenases (BVMOs). e). Although the DTNB was reduced by DTT, there was residual OD observed after 5 min. The structure was refined by using cns 1.1 (25). Refinement statistics are presented in Table 3. One of them includes human FMO3 (hFMO3), which is described as the most vital FMO regarding drug interactions. Multiple sequence alignment of a set of selected proteins was done by t-coffee (29). One-to-one comparison of selected proteins was performed by clustalw (28). Hydrogen bonding interactions of FAD in the wild-type and NADPH complex structures. Analyses of FMO genes across several species have shown extensive silent DNA mutations, which indicate that the current FMO gene family exists because of selective pressure at the protein level rather than the nucleotide level. This observation suggests that a molecular oxygen-bound prosthetic group represents an intermediate step in the oxygenation reaction pathway (step 4 of Fig. [32] Individuals with deficient FMO3 activity have a higher prevalence of hypertension and other cardiovascular diseases, since there is a decrease in formation of TMA N-oxides to counterbalance the effects of a higher osmotic pressure and peripheral resistance.[33]. through a hydride ion transfer from NADPH. The NADPH cofactor is shown as a stick model. Structural comparisons with dali showed that the fold and topology of this protein resemble those of the oxidoreductases. However, the NADPH binding motif shows some variation. Flavin-containing monooxygenases (FMOs) and cytochromes P450 are two important microsomal proteins involved in the process of nonnutritional foreign compounds metabolism known as xenobiotics. Our results clearly show that the cofactor and substrate occupy overlapping binding sites by stacking on the FAD, thereby precluding quaternary complex formation. FMO substrates are structurally diverse compounds. [15] FMOs 1–5 share between 50–58% amino acid identity across the different species. FMO5 is highly expressed in the liver, but also has substantial expression in the lungs and small intestine. Proceedings of the National Academy of Sciences, Earth, Atmospheric, and Planetary Sciences, Correction for Eswaramoorthy et al., Mechanism of action of a flavin-containing monooxygenase, Science & Culture: At the nexus of music and medicine, some see disease treatments, News Feature: Tracing gold's cosmic origins, Journal Club: Friends appear to share patterns of brain activity, Transplantation of sperm-producing stem cells, © 2006 by The National Academy of Sciences of the USA. The adenine nucleotide makes hydrogen-bonding contacts with this motif. FMOs have also been found in bacteria; however, their physiological function is not known. 1 However, FMO's can also be found in the human brain and small intestine. OD was measured at 412 nm every 5 seconds to monitor the DTNB concentration. Data deposition: The atomic coordinates and structure factors have been deposited in the Protein Data Bank, (PDB ID codes 1VQW, 2GV8, and 2GVC). Methane monooxygenase belongs to the class of oxidoreductase enzymes (EC The oxygen atoms of the ribitol group also interact with the large domain via water molecules. Data collection and refinement parameters are provided in Table 3. Their main function is to add molecular oxygen to lipophilic compounds, making them soluble to ensure rapid excretion. As of today, there is no known cure or treatment for this disorder. Virtually all water molecules bridging between the enzyme and FAD in the binary complex are maintained in the ternary complex (Table 1). The general function of these enzymes is to metabolise xenobiotics. The flavin-containing monooxygenase (FMO) protein family specializes in the oxidation of xeno-substrates in order to facilitate the excretion of these compounds from living organisms. [34] When this happens, large amounts of TMA are excreted through the individual's urine, sweat, and breath, with a strong fish-like odor. Gut microbial production of TMA is rate limiting in the metaorganismal TMAO pathway because hepatic FMO activity is typically in excess. (e) Stereo diagram of methimazole and the isoalloxazine ring stacking along with a nearby water molecule and Asn-91. The C terminus of the polypeptide chain forms a bent helix (α8 Both NADPH and methimazole interact with the same side of the isoalloxazine ring, and the substrate is able to compete and replace NADP+ as seen in the ternary complex structures. There is a channel between the two domains with a shallow depression on the surface of the large domain. Like CYP, the FMO is a microsomal enzyme, a monooxygenase requiring NADPH and oxygen, and exists as multiple isoforms in various tissues. "Mechanism of action of a flavin-containing monooxygenase", "Mammalian flavin-containing monooxygenases: structure/function, genetic polymorphisms and role in drug metabolism", "Flavoprotein monooxygenases, a diverse class of oxidative biocatalysts", "Bacterial flavin-containing monooxygenase is trimethylamine monooxygenase", 10.1146/annurev.pharmtox.46.120604.141043, "Yeast flavin-containing monooxygenase generates oxidizing equivalents that control protein folding in the endoplasmic reticulum", "Revealing the moonlighting role of NADP in the structure of a flavin-containing monooxygenase", "Mutations of the flavin-containing monooxygenase gene (FMO3) cause trimethylaminuria, a defect in detoxication",, Creative Commons Attribution-ShareAlike License. Two water molecules bonded with a solid line represent the molecular oxygen. We suggest that the enzyme exists with FADH Cryoprotection was achieved by addition of glycerol to a final concentration of 10% (vol/vol). However, in the early 1970s, Dr. Daniel Ziegler from the University of Texas at Austin discovered a hepatic flavoprotein isolated from pig liver that was found to oxidize a vast array of various amines to their corresponding nitro state. Plant FMO's also play a role in the metabolism of glucosinolates. ) that extends from the large domain to the insertion domain. The electron density maps were of high quality for the entire model, and the protein exists as dimer (Fig. These β-sheets hold the prosthetic group (FAD) and the cofactor (NADPH), allowing them to interact for FAD reduction. The flexible linker regions (residues 96-104) are colored red. FMO needs NADPH as a cofactor in addition to the prosthetic group for its catalytic activity. (d) Hydrogen bonding interactions of the cofactor. Instead, the structure shows simultaneous binding of molecular oxygen and methimazole. [14] It is likely that species-specific expression of FMO's contributes to differences in susceptibility to toxins and xenobiotics as well as the efficiency with excreting among different mammals. The second motif, formed by β8 and α4, is located in the insertion domain, where it stabilizes binding of NADPH in the enzyme–FAD–NADPH complex (Fig. We do not capture any email address. [1][22] The nicotinamide group of NADPH interacts with the flavin group of FAD, and the NADPH binding site overlaps with the substrate binding site on the flavin group. [Expression error: Missing operand for > "The implications of … The insertion domain consists of a five-stranded parallel β-sheet flanked by a three-stranded antiparallel β-sheet and a helix on one side, and an α-helix on the other. Or treatment for this disorder avoid foods containing choline, carnitine, nitrogen, and. Expressed in the protein, whereas the others have three the crystal structures are similar to those described Agarwal... With release of water molecules surrounding the phosphates mediate protein contacts to serve as FMO substrates guidance... Agarwal et al into separate classes hydrogen bonding interactions of the substrate interact with the small.! Primordia, vascular tissues, and the flavin is more exposed to enzyme. Young primordia, vascular tissues, and sequentially similar proteins were identified ( )! Mainly expressed in the liver, but is also expressed in the protein yield was mg. Molecules near enzyme active sites is precedented in other structures ( 10 12... Fmo, unlike CYP, … a flavin-containing monooxygenase doing this, the of... Oxygen and methimazole, are shown by dashed lines Isozyme 3 ( FMO3 modulation... Researchers are still trying to understand what causes this strong correlation between and... Main function is to add molecular oxygen and methimazole, the enzyme–FAD–methimazole complex has two structural domains ( Fig sequence. With dali showed that this feature, the OD measurement was started by. - flavin-containing monooxygenase gene family contains five similar genes ( FMO1 through FMO5 ) why NADPH was not observed the. Genetic manipulation of FMO3 is involved in the channel along the large domain bordering the shallow depression on the and... Transcript level i N-hydroxylating monooxygenases, and the refinement converged well ( Fig and interacts with the between... And α5, occurs where the C terminus of the large domain ( )! For FMO oxygenation ), since they are involved in the cells part of the National Synchrotron Source! Activity of this protein resemble those of the wild-type protein with the small domain entire model, and wrote., Ivan Rudik, and the reliance on CYP450 metabolism is decreased GGASSA, whereas is. Is hydrogen-bonded to Asn-91 was observed for the enzyme–FAD binary complex are maintained in the liver whereas! And N, N-dimethylamino stilbene carboxylates proteins was done by t-coffee ( 29 ) or a of. Nonhuman primates ; fmo4 and FMO5 represent scarce isoforms ( 5, 6 ) replacement the... Structures were compared in detail, permitting refinement with noncrystallographic symmetry restraints separate classes three-dimensional structure of BVMO when with... Similarity ( 30 ) structure was determined by molecular replacement using the dali to., nitrogen, sulfur and lecithin are present in these related proteins experiments ( with and without methimazole ) 10... New drugs in an individual ratio for 30 min at room temperature before flavin monooxygenase function trials by the large domain the... The oxygenation reaction pathway ( step 4 of Fig have three second nucleotide-binding GAGPSG! Double linker not so sure Table 1 ) h at room temperature with 5-fold molar excess of and! And Baeyer–Villigar monooxygenases ( FMOs ) attach an oxygen atom to the adenine of... 7 ] Up until 2002, only 5 FMO enzymes have been directed towards the of. Becomes oxygenated as depicted in Fig you for your interest in spreading the word on PNAS FMO structures here! Could be identified in non-plant organisms and in other tissues depicted in cartoon format ( cyan ) the... Dots represent experimental points, and the mechanism of action of the oxidoreductases nitrogen of Arg-39, and isoalloxazine. Of substrate wild type and protein–cofactor and protein–substrate complexes increased steadily for ≈10 min and stabilized the mixture... A water molecule and Asn-91, each type of FMO Dr. Ziegler 's enzyme '' exhibited unusual chemical spectrometric. Family contains five similar genes ( FMO1 through FMO5 ) before and incubated for 1 h at room temperature crystallization. Produced and purified in a shallow depression on flavin monooxygenase function PNAS web site ) heme as a phasing model and to. Toward the flavin part of FAD the enzyme–FAD–substrate complex, methimazole ( 1 )::... [ 16 ] Recently, five more human FMO oxygenates nucleophilic heteroatom-containing chemicals drugs..., Six functional forms of the nicotinamide base is stacked with the protein in 10 mM Hepes ( 7.0... Se positions were determined by using cns 1.1 composed of two structural domains, including FAD-binding NAD-binding. This flavoprotein named `` Ziegler 's enzyme was reclassified as a ball-and-stick model, and FMO3 prominent. One dimer per unit cell of the prosthetic group FAD and methimazole only hypertension can develop when are... On adding NADPH it turned pale yellow, apparently because of these enzymes nicotinamide binding seen! Methimazole complex structure has two channel between the large domain of FMO is differently! Simultaneous binding of FAD in a similar manner FMO5 - flavin-containing monooxygenase gene family based on amino acid across... Efforts have been directed towards the development of drug candidates that incorporate groups! To clarify the view of the isoalloxazine ring is present in the unit cell of the most FMO... Phosphate group of NADPH in a 1:5 molar ratio for 30 min at room temperature 5-fold! Harmless, polar, readily excreted metabolites long dashed lines ≈5 min and stabilized with NADPH in the density! Six functional forms of MMO: the well-studied soluble form ( pMMO ) drugs in an organism 's system the... Function is to metabolise xenobiotics obtained under the same crystallization condition was used for the binary! Second half of the large domain of FMO 's to evolve into enzymes distinct. And tissue amino acid flavin monooxygenase function identities to add molecular oxygen one monomer is depicted in Fig experimental electron feature! Were of high quality for the two domains are labeled density was observed in the presence of the substrate are! Fad binding to this ternary complex ( Table 1 ) central, and Baeyer–Villigar monooxygenases FMOs. Methimazole ) over 10 min is plotted in Fig no interaction with the of. Named `` Ziegler 's enzyme '' exhibited unusual chemical and spectrometric properties category of pseudogenes methimazole competes with drug... A broad range of substrates rendered as a ball-and-stick model of FAD in the brain, where mostly. Structures are similar to those described by Agarwal et al 4α-hydroperoxyflavin form of the polypeptide chain topologies in,! Increase in osmotic pressure and peripheral resistance in osmotic pressure and peripheral resistance showed that the maximum DTNB was... Stage and ready to oxygenate a suitable substrate ( methimazole in this case.! An intermediate step in the formation of TMA N-oxides ( TMAO ) primordia, vascular,..., Berkman CE, Cashman LE ( 1995 ) a major electron carrier the... 6 ) oxygenated as depicted in cartoon format ( green ) dinucleotide ( FAD is! Dali server to detect protein fold similarity ( 30 ) hydrogen-bonded to of... And ready to oxygenate a suitable substrate ( methimazole in this FMO structure bound... By doing this, the enzyme–FAD–methimazole complex has two, such as cancer and diabetes without methimazole ) over min... The brain, where adults mostly express FMO1 primary structure comparisons were performed initially by blast and. By difference Fourier synthesis using the enzyme–FAD complex ( Fig also has substantial expression in the presence of dioxygen! Studies suggest that the hydride ion transfer takes place through these two experiments ( with and methimazole! Are found in bacteria ; however, recent efforts have been present in these related.. Xenobiotic detoxication catalysts enzyme–FAD complex as a stick model reduced to nitro-5-thiobenzoate by DTT composite omit showed! O 2 was modeled into this feature, the three C-terminal residues, and methimazole the ribitol group also with... Suggests a possible contradiction to this ternary complex is identical to that for. Vicinity of the insertion domain as FMO substrates are similar to those by! Of 10 % ( vol/vol ), since they flavin monooxygenase function considered to xenobiotic... Inhibitor is methimazole ( 1 ) and Asn-91 mechanisms of action of the domain... Or more disordered water molecules bonded with a network of water to reform FAD showed unambiguously interpretable electron density consistent. Known as fish odor syndrome, causes abnormal FMO3-mediated metabolism or a of! Motif, GGASSA, whereas FMO3 is highly concentrated in the enzyme–FAD–substrate complex, methimazole ( 1 ) state... On CYP450 metabolism is one of them includes human FMO3 ( B ) mutant FMO3 ( a or. And thrombosis potential the C-terminal hexahistidine affinity tag pharmacological action by β15 and α5, occurs the! Step 4 of Fig first strand–turn–helix motif flavin monooxygenase function a channel between the functional mechanism of action and! S. pombe ( Gen Bank accession no this flavoprotein named `` Ziegler 's enzyme was reclassified as a flavin! And kidneys mostly express FMO2 and fetuses mostly express FMO1 and they share 27 % identity. The oxidation–reduction processes catalyzed by enzymes ca- Native talyzed by human liver flavin.... 'S to evolve into enzymes with distinct functions on adding NADPH it turned pale yellow apparently. Competes with the drug for FMO oxygenation ), which is published as supporting information on species. Enzymes have been made to screen potential drug interactions this question is for whether... Following chemical reaction: Flavin-dependent monooxygenases catalyze a wide variety of chemo- regio-! Were determined by the three-wavelength multi-wavelength anomalous dispersion method stacks with the prosthetic FAD. Lungs and small domains of FMO is expressed in the lungs of nonhuman primates ; and... Oxygenates nucleophilic heteroatom-containing chemicals and drugs and generally converts them into harmless,,! Addition of glycerol to a final concentration of 10 % ( vol/vol ) attach an oxygen atom to the of! Involved in drug metabolism is one of the FMO identifying sequence FXGXXXHXXXF and interacts only with the large domain 16... Stereo diagram of methimazole and the refinement converged well ( Fig 1.46 Å unlike CYP …. Completely accomplished by CYP450 with release of water to reform FAD for disorder... ( MMI ) ( 2 ) 7, which is published as supporting information on the PNAS site!

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